In recent years, it has become possible to easily generate genetically modified animals via microinjection of artificial nucleases ZFNs/TALENs/CRISPRs into fertilized eggs in mammals, including mice and rats. Artificial nucleases introduce DNA double-strand breaks (DSBs) into target genes, and indel mutations are introduced via non-homologous end joining (NHEJ), which is a DSB repair mechanism, thereby generating knockout animals in which the target genes are disrupted. Since knockout animals can be generated more efficiently at lower cost in a shorter period of time than conventional gene modification techniques using ES cells, this technique has been widely used as a technique for generating genetically modified animals (NPL 1 and 2).
Attempts for knock-in have also been made to introduce a gene, such as GFP, into a target genomic region (or gene) using an artificial nuclease. A donor plasmid having an about 500 bp to 1 kbp sequence homologous to a target genomic region at each end of the knock-in sequence of GFP or the like is used. By introducing the donor plasmid into fertilized eggs together with an artificial nuclease, the artificial nuclease introduces DSB in the target sequence, and the gene, such as GFP, is knocked-in into the target sequence using the homologous sequences of the donor plasmid via homologous recombination (HR), which is another DSB repair mechanism (NPL 3 and 4).
In a method using no donor plasmid, a single base in a target gene can be substituted or a short DNA sequence of His-tag, LoxP, etc., which has a length of not greater than tens of bp, can be introduced, using single-stranded DNAs (ssODNs: single-stranded oligodeoxynucleotides). Knock-in animals can be generated easily and efficiently using single-strand annealing (SSA), which is a highly efficient DSB repair mechanism, by artificially synthesizing an ssODN comprising a 40- to 60-bp homologous sequence at each end of a base sequence to be introduced, and introducing the ssODN into fertilized eggs with an artificial nuclease (NPL 5 and 6).
When knock-in is performed using a donor plasmid, it is necessary to add, to a plasmid containing a gene to be knocked-in, sequences homologous to a target genomic region. Conventionally, such homologous sequences are amplified, for example, by PCR, ligated, and cloned in Escherichia coli to produce a donor plasmid, which takes time and effort. Moreover, it has been reported that HR is generally far less efficient than NHEJ in DSB repair for mammalian cells or fertilized eggs. Even when a donor plasmid is microinjected with an artificial nuclease, the efficiency of generating knock-in animals is notably lower than the efficiency of generating knockout animals (NPL 3 and 7).
When using ssODNs, knock-in is performed via DSB repair mechanism SSA using a single-stranded DNA, which is different from HR. It has thus been reported that the efficiency is higher than that when a donor plasmid is used (NPL 5 and 6). However, since only ssODNs of up to about 200 bp in length can be accurately synthesized, it is difficult to knock-in a long-chain gene sequence of GFP or the like (several hundred bp to several kilo bp).